Methods and Kits for Measuring Toxicity and Oxidative Stress in Live Cells

ABSTRACT

Compositions and methods for the measuring toxicity, antioxidant capacity, and oxidative stress are provided.

This application is a divisional application of U.S. patent applicationSer. No. 13/320,602, filed on Feb. 1, 2012, which is a national stageapplication of PCT/US2010/034899, filed on May 14, 2010, which claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationNo. 61/178,929, filed on May 15, 2009; U.S. Provisional PatentApplication No. 61/261,521, filed on Nov. 16, 2009; and U.S. ProvisionalPatent Application No. 61/290,084, filed on Dec. 24, 2009. Each of theforegoing applications is incorporated by reference herein.

This invention was made with government support under Grant NumberCA109604 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to oxidative stress, cytotoxicity, andantioxidants. More specifically, assays for measuring oxidative stressand cytotoxicity and methods of use thereof are provided.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limitingenzyme of the oxidative pentose phosphate cycle (OPPC). Glucose, asubstrate for the OPPC, is required for OPPC mediated detoxification ofoxidants/disulfides. Glucose is utilized as a substrate by oxidativepentose phosphate cycle to generate reductants. These reductants areutilized to maintain reduced glutathione homeostasis in mammalian cellswhen exposed to oxidants/disulfides. Glutathione is a tripeptideconsisting of glycine, cysteine and glutamate. The reduced glutathione(GSH) is up to 100 folds higher than the oxidized GSH (GSSG) inmammalian cells under normal conditions.

Oxidative stress is presently quantified by measuring the ratio ofreduced glutathione (GSH) to oxidized glutathione (GSSG). Oxidizedglutathione is the most commonly used biomarker in biomedical research.However, the various biochemical assays currently available require thepreparation of tissue extracts and cannot be applied to humans becauseof their invasiveness. Indeed, while HPLC with electrochemical detectioncan monitor glutathione with better sensitivity than other biochemicalassays, the method still requires tissue or cellular extracts.

Furthermore, these assays may overestimate the extent of oxidativestress since depletion of GSH measured by biochemical assays may alsoinclude oxidation of GSH during lysis of cells and extract preparation.Moreover, none of these assays measures the function of GSH in livecells. In view of the foregoing, it is evident that there is a need forimproved oxidative stress assays in live cells.

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, methods ofmeasuring oxidative stress in a live cell are provided. In a particularembodiment, the methods comprise contacting cells withhydroxyethyldisulfide (HEDS) and determining the amount of extracellularmercaptoethanol, wherein the amount of extracellular mercaptoethanol isinversely proportional to the oxidative stress of the cell. In anotherembodiment, the amount of extracellular mercaptoethanol is directlyproportional to the glutathione recycling capacity of the cell.

According to another aspect of the instant invention, methods ofscreening for an antioxidant are provided. In one embodiment, themethods comprise a) contacting a first cell with hydroxyethyldisulfide(HEDS); b) determining the amount of extracellular mercaptoethanolproduced by the first cell; c) contacting a second cell with a compound;d) contacting the second cell with hydroxyethyldisulfide (HEDS); and e)determining the amount of extracellular mercaptoethanol produced by thesecond cell; wherein the presence of more extracellular mercaptoethanolfrom the second cell indicates that the compound is an antioxidant. Instill another embodiment, the method comprises a) contacting a cell witha compound; b) contacting the cell with hydroxyethyldisulfide (HEDS);and c) determining the amount of extracellular mercaptoethanol producedby the cell, wherein the presence of more extracellular mercaptoethanolfrom the cell compared to a standard indicates that the compound is anantioxidant. In another embodiment, the first and second cells are frombiological samples and the compound is administered to a subject.

In yet another aspect, methods of measuring the cytotoxicity of acompound are provided. The methods may comprise contacting cells withthe compound, contacting the cells with hydroxyethyldisulfide (HEDS),and determining the amount of extracellular mercaptoethanol for thecells; wherein a decrease in the amount of extracellular mercaptoethanolfor the cells compared to cells not exposed to the compound indicatesthat the compound is cytotoxic. In a particular embodiment, the methodcomprises obtaining a biological sample from a subject, administeringthe compound to the subject, and obtaining a second biological samplefrom the patient. The cells of the first and second biological samplesare then contacted with hydroxyethyldisulfide (HEDS) and the amount ofextracellular mercaptoethanol is determined.

According to still another aspect, kits for practicing the methods ofthe instant invention are provided. In a particular embodiment, the kitscomprise a) 5,5-dithiobis 2-nitrobenzoic acid (DTNB); b)hydroxyethyldisulfide (HEDS); and c) sulfosalicyclic acid buffer (SSA).The kits may also comprise at least one of: DTNB buffer, HEDS buffer,96-well plate(s), and reaction buffers containing glucose-6-phosphatedehydrogenase, thioredoxin reductase, thioredoxin or other antioxidants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B demonstrate the conversion of HEDS into mercaptoethanol(ME) by human cells as determined by High Performance LiquidChromatography (HPLC)/electrochemical analysis. FIGS. 1A and 1B are HPLCchromatograms obtained from the extracellular medium after 2 hoursincubation of human cells with (FIG. 1B) and without (FIG. 1A) HEDS.FIG. 1A shows only a solvent peak (peak 1) for extracellular medium fromcells incubated for 2 hours without HEDS. FIG. 1B shows a solvent peak(peak 1), a mercaptoethanol (ME) peak (peak 2), and a HEDS peak (peak 3)for extracellular medium from cells incubated for 2 hours with HEDS.

FIGS. 2A and 2B depict the measurement of arsenite induced oxidativestress in human cells in vitro as measured by HPLC/electrochemicalanalysis of extracellular medium samples. HPLC chromatograms for humancells incubated with 0 (FIG. 2A) and 10 μM (FIG. 2B) arsenite for 24hours in vitro are provided. FIGS. 2A and 2B show a solvent peak (peak1), a ME peak (peak 2), and a HEDS peak (peak 3) for extracellularmedium from human cells incubated with HEDS for 2 hours after 24 hourstreatment with and without arsenite. The results show that 10 μMarsenite (FIG. 2B) decreased the ME peak (peak 2) by almost 70% with acorresponding increase in the HEDS peak (peak 3) compared to the cellsnot treated with arsenite (FIG. 2A). Chromatograms without HEDS forthese samples showed only a solvent peak.

FIG. 3 is a graph of HEDS conversion measured by the HEDS/DTNB assay ofthe instant invention of the extracellular medium from human HCT116cells treated with different concentrations of arsenite. The results inFIG. 3 show an arsenite concentration dependent decrease in HEDSconversion by human cells incubated with arsenite for 24 hours. HEDSconversion was measured immediately after the 24 hours incubation ofcells with arsenite.

FIG. 4 is a graph of arsenite toxicity measured by Coulter CounterAnalysis of cells harvested six days after arsenite treatment. Theresults in FIG. 4 demonstrate an arsenite concentration dependentdecrease in the survival of human HCT116 cells incubated with arsenitefor 24 hours. The cell growth was measured six days after removing thearsenite from the extracellular medium by washing the cells three timeswith fresh growth medium.

FIGS. 5A-5C are graphs of the application of the assay of the instantinvention compared to other commercially available assays (WST-1 andXTT) used for cell survival in tissue culture medium. The figure shows alinear cell density dependent conversion of HEDS better than thecommercially available assays (WST-1 and XTT) measured 16 hours afterplating the cells in a 96 well microtiter plate. Cells tested: HCT116cells (FIG. 5A), MCF10A cells (FIG. 5B), and MCF7 cells (FIG. 5C).

FIG. 6 is a graph showing the application of the assay of the instantassay to quantify the toxicity of cisplatin in human cells in a 96 wellplatform. FIG. 6 demonstrates that the instant assay can be used tomeasure the toxicity induced by cisplatin in human cells. The resultsshow a ciplatin dose dependent decrease in HEDS conversion measured 5days after overnight incubation of cells with micromolar (0, 10, 20, 30,40, or 50 μM) concentrations of cisplatin.

FIGS. 7A-7C are graphs of the total antioxidant capacity of various celldensities of three cell lines: Jurkat (FIG. 7A), CCRF-CEM (FIG. 7B), andT98G (FIG. 7C).

DETAILED DESCRIPTION OF THE INVENTION

Glucose is converted into glucose-6-phosphate by hexokinase.Glucose-6-phosphate is used as a substrate by G6PD/oxidative pentosephosphate cycle to produce NADPH. NADPH is used as a cofactor to reducedisulfides into monosulfides, which are released into the medium. Thisconversion is dependent on the glucose level and active metabolicpathway of live cells.

In a particular embodiment, the assay of the instant invention usehydroxyethyldisulfide (HEDS), a unique non-toxic disulfide, to measureglucose dependent metabolic activity of live cells in vitro. Thesurvival of cells is directly proportional to the conversion of HEDSinto mercaptoethanol (ME) by metabolically active live cells. Dead cellsfail to convert HEDS into mercaptoethanol. Accordingly, the assays ofthe instant invention generally encompass contacting cells (e.g., livecells) with HEDS and then monitoring or measuring the amount ofextracellular ME. The amount of ME produced from HEDS can be measured inthe extracellular medium since ME is extruded out quickly into theextracellular medium. Extracellular ME may be measured, for example, bya dithiobiznitrobenzoic acid (DTNB) assay or High Performance LiquidChromatography/Electrochemical Detection (HPLC/EC). The cells of theassay may be any cell that produces NADPH, particularly mammalian cellsand yeast. The percentage of live cells may be determined by the amountof ME produced.

The general assay of the instant invention may be used in a variety ofmethods. For example, the assay may be used 1) to screen for oxidativestress in live cells, 2) as a cell survival assay, 3) to screen for thetoxicity of a compound, 4) to screen for exposure to or the presence ofa toxin such as arsenical compounds, 5) to screen for toxicity or stressin subjects treated with cancer chemotherapeutic agents and/orradiation, 6) to measure glutathione recycling, 7) to screen theefficacy of an antioxidant or composition, 8) high throughput screeningof chemicals/drugs that target biological molecules involved in cellsurvival, 9) to screen for tumor cell resistance to radiation and/orchemotherapeutic reagents, and 10) to predict radiation and/orchemotherapeutic response using blood samples from a subject. Thesemethods are described in more detail below.

In accordance with one aspect of the instant invention, methods formeasuring oxidative stress in a live cell are provided. The methodcomprises contacting the cells with HEDS and subsequently measuring theamount of extracellular ME, wherein the amount of extracellular ME isinversely proportional to oxidative stress. The method may furthercomprise obtaining a biological sample comprising the cells from asubject. In a particular embodiment, the method comprises obtaining atleast one biological sample comprising cells from a subject,administering at least one compound to the subject, obtaining at leastone second biological sample from the subject, and determining theamount of extracellular ME after HEDS administration. An increase in theextracellular ME from the second biological sample indicates that the atleast one compound reduces oxidative stress. Additionally, while theinstant methods comprise obtaining a first and second biological sample,the instant invention also encompasses a method wherein only at leastone biological sample(s) are obtained after administration of thecompound and the amount of extracellular ME after HEDS administration iscompared to a standard(s) (e.g., the amount of extracellular ME afterHEDS administration in a biological sample from a subject(s) notadministered the compound). While the method is exemplified above withadministering at least one compound to the subject, the method may alsobe used to measure toxicity after any challenge (e.g., after treatmentwith/exposure to radiation, chemotherapeutic agents, cytotoxins, and thelike).

In accordance with another aspect of the instant invention, methods toquantify the survival/viability of cells are provided. As stated herein,cell death is directly proportional to the lack of conversion of HEDSinto ME. In a particular embodiment, the method comprises contacting thecells with HEDS and subsequently measuring the amount of extracellularME, wherein the amount of extracellular ME is proportional to cellviability. Optionally, a standard curve of extracellular ME productionto viable cell number can be generated prior to testing an unknownsample.

Currently, there are four major assays commonly used as celldeath/survival assays. However, there are substantial problems withthese cell viability and cytotoxic assays. The cell survival assay ofthe instant invention overcomes the problems associated with theseassays.

The first major assay is the lactose dehydrogenase (LDH) assay. Thisassay measures the release of LDH after membrane damage caused bycytotoxic drugs. One problem with this assay is that it requirescollection of extra cellular medium at different time points since it isnot known at what time the membrane leak occurs after cytotoxictreatment. The assay may also not be sensitive enough to detect lowlevel of cell death. Further, most cytotoxins kill cells byintracellular mechanisms without damaging the plasma membrane and theassay only works for cytotoxins that induce high cell lysis within ashort time. There is also a need to determine the peak response timeafter treatment for this assay and the peak response may vary atdifferent concentrations of cytotoxins. Lastly, cells need to be grownin special medium without low serum since LDH in the serum willinterfere with this assay.

A second major assay is the glucose consumption dependent H₂O₂ assay.This assay measures glucose consumption, which is proportional to thenumber of cells. The problem with this assay is that most cells consumeglucose at a very slow rate and most medium has 25 mM glucose. If themedium has high concentration of glucose, there is a need to dilute themedium and errors may occur.

The third major assay is the(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT)assay. Purple formazan is formed by mitochondrial reduction of MTT inliving cells. MTT, however, is not water-soluble and needs to besolubilized by DMSO. Furthermore, there may be interference from themedium, especially if the medium color changes when cells are confluentor when there is a change in pH of the medium.

Lastly, the fourth major assay is the sodium3″-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitrobenzene sulfonic acid hydrate (XTT). A colored formazon product isformed by reduction of XTT. As with the MTT assay, there may beinterference from the medium if the medium color changes when cells areconfluent or there is a change in the pH of the medium. The XTT assayalso requires phenazine methosulfate (PMS) to effectively reduce XTT.However, PMS is a known generator of oxygen free radicals, which causespotassium loss in cells. The reduction of XTT is solely dependent onNADH. More specifically, the reduction of XTT is caused by NADH releasedinto the extracellular medium through trans-plasma membrane electrontransport. The amount of extracellular reduction is not as effective asintracellular bioreduction. Furthermore, studies in yeast had shown thatseveral factors such as iron and membrane enzymes other than NADH mayalso be responsible for bioreduction of XTT, thereby interfering withthe assay.

In accordance with another aspect of the instant invention, methods formeasuring the cytotoxicity of at least one compound are provided. In aparticular embodiment, the method comprises providing at least twopopulations of cells wherein one population of cells has been contactedwith the at least one compound and a second population of cells as acontrol which has not been contacted with the at least one compound. Themethod further comprises contacting the populations of cells with HEDSand subsequently measuring the amount of extracellular ME. A reductionin the extracellular ME from the population of cells contacted with theat least compound indicates that the at least one compound is cytotoxic.While the method is exemplified above with contacting the cells with atleast one compound, the method may also be used to measure viabilityafter any challenge (e.g., after treatment with radiation,chemotherapeutic agents, cytotoxins, and the like). The method may beused with high throughput screening of compounds. In a particularembodiment, the compound is a drug such as chemotherapeutics oranti-yeast, fungal or bacterial drugs.

In accordance with another aspect of the instant invention, the assay ofthe instant invention is used for high throughput screening ofchemicals/drugs that target glucose 6-phosphate dehydrogenase,thioredoxin reductase, thioredoxin, or other antioxidants. In oneembodiment, the method comprises contacting purified glucose 6-phosphatedehydrogenase, thioredoxin reductase, thioredoxin, or other antioxidantswith HEDS and subsequently measuring the amount of extracellular ME in areaction mixture. In a particular embodiment, the method comprisesproviding at least one compound selected from the group consisting ofglucose 6-phosphate dehydrogenase, thioredoxin reductase, thioredoxin,and other antioxidant, in a reaction mixture (e.g., in a 96-well plate)wherein this reaction mixture has been contacted with at least one testcompound (e.g., from a chemical library). The method further comprisescontacting this mixture with HEDS and subsequently measuring the amountof ME. A decrease in ME indicates that the at least one compoundpossesses inhibitory effect on the target molecules such asglucose-6-phosphate dehydrogenase, thioredoxin reductase, thioredoxin,or other antioxidants.

In another embodiment of the instant aspect, the method comprisesobtaining a biological sample comprising cells from a subject,administering at least one compound to the subject, obtaining a secondbiological sample from the subject, and determining the amount ofextracellular ME after HEDS administration. A reduction in theextracellular ME from the second biological sample indicates that the atleast one compound is cytotoxic. As stated hereinabove, the method mayalternatively comprise obtaining at least one biological sample afteradministration of the compound and comparing to a standard(s). While themethod is exemplified above with administering at least one compound tothe subject, the method may also be used to measure toxicity after anychallenge (e.g., after treatment/exposure with radiation,chemotherapeutic agents, cytotoxins, and the like).

In yet another embodiment of the instant aspect, the method may be usedto screen for compounds which promote or increase cell survival. Forexample, the method may comprise contacting one of the populations ofcells with at least one compound and, optionally, maintaining the cellsunder conditions which cause cell death, and then measuringextracellular ME after HEDS administration.

In accordance with another aspect of the instant invention, methods ofdetecting exposure to or the presence of a toxin, particularly anoxidative stress inducing toxin, are provided. In a particularembodiment, the method comprises contacting cells with HEDS andsubsequently measuring the amount of extracellular ME. The method maycomprise obtaining a biological sample comprising cells from a subject,optionally a subject suspected of having been exposed to the toxin. In aparticular embodiment, the method comprises obtaining an environmentalsample (e.g., a water and/or soil sample) and contacting the sample withHEDS and subsequently measuring the amount of ME. In still anotherembodiment, cells are contacted (e.g., cultured) with the environmentalsample prior to contacting with HEDS. The lack of HEDS conversion in theinstant assays correlates (e.g., indicates an increased risk/likelihood)with toxin (e.g., arsenical, radiation, chemotherapeutic agent, etc.)induced GSH depletion and cell death. Thus, the inhibition of HEDSconversion is an effective biomarker of oxidative stress that can beused to detect (or indicate an increased risk for) exposure to or thepresence of toxins such as arsenicals. The method may also comprisecomparing the extracellular ME to a standard (e.g., a biological sampleobtained from a subject known not have been exposed to the toxin orexposed to a known toxin; or other samples (e.g., environmental sample)comprising a known amount of the toxin). In a particular embodiment, thetoxin is an arsenical compound, pollutant, mercury, cadmium, lead,asbestos, barium, carbon tetrachloride, chloromethane, chromium, copper,dichloroethene (DCE), polychlorinated biphenyls (PCBs), polycyclicaromatic hydrocarbons (PAHs), selenium, silver, sulfuric acid,tetrachloroethene (PCE), trichloroethane (TCA), trichloroethene (TCE),vinyl chloride, zinc, acetaldehyde, acetonitrile, acrylamide, acrylicacid, aniline, 1,1′-biphenyl, 1-butanol, butraldehyde, carbon disulfide,carbonyl sulfide, chlorine, chlorobenzene, cyclohexane, 1,4-dioxane,freon 113, methanol, methyl ethyl ketone, methyl isobutyl ketone, methylmethacrylate, methyl-tert-butyl ether, methylchloroform(1,1,1-Trichloroethane), methylene chloride (dichloromethane),2-methoxyethanol, nitrobenzene, perchloroethylene, phthalic anhydride,styrene, toluene, trichlorobenzene, or 1,2,4-trimethylbenzene. In aparticular embodiment, the toxin is an arsenical compound.

Arsenicals are the most common contaminants in soil, ground water, food,and plants. More than 15 million people in the U.S. and around 100million in the rest of the world are likely to be exposed to arsenicals.Arsenicals are known to cause oxidative stress, which may be responsiblefor the toxic, carcinogenic, and mutagenic effects of arsenicals inhumans and animals. Arsenicals are also known to cause atherosclerosis,cancer, and other diseases in humans. Inorganic arsenic commonly existsas arsenate (As⁵⁺ and arsenite (As³⁺) in ground water. Oxidation of As³⁺to As⁵⁺ may increase the As⁵⁺ content in the soil. The semi-metallicform of arsenic oxidizes rapidly in air, and at high temperaturesproduces arsenic trioxide. In addition, arsenic trioxide is currentlyused in humans to treat cancer. Phenylic arsenic compounds are the maincontaminants in groundwater at abandoned sites with a history of arseniccontaining chemical weapon agents. Exemplary arsenicals include, withoutlimitation, sodium arsenate, sodium arsenite, arsenic trioxide andphenylarsine oxide, as these arsenicals are environmental pollutants.These arsenicals can cause oxidative stress leading to lipidperoxidation, protein oxidation, and depletion of glutathione (GSH) andother antioxidants in mammalian cells. Herein, it has been determinedthat the toxicity of arsenic compounds is dependent on the extent ofinhibition of HEDS conversion in cells.

In accordance with another aspect of the instant invention, methods formeasuring the glutathione recycling capacity of a live cell areprovided. The method comprises contacting the live cells with HEDS andsubsequently measuring the amount of extracellular ME. It has beendemonstrated that glutathione (GSH) recycling (GSH→GSSG→GSH) isimportant for cellular defense against oxidative stress. An assay thatmeasures the capacity of cells to recycle glutathione, rather than justmeasuring GSH and GSSG, is more appropriate in determining metabolicoxidative stress since GSH recycling covers the total metabolicoxidative stress. The conversion of HEDS requires GSH recycling byconverting GSSG, which is produced during HEDS conversion, back to GSH.Defects in GSH recycling in cells affect the conversion of HEDS intomercaptoethanol. The assay of the instant invention is not only usefulfor laboratory based biomedical research (e.g., tissue culture andanimals) but also for use on subjects.

In accordance with another aspect of the instant invention, the assay ofthe instant invention can be used to investigate the antioxidantproperty of at least one compound (e.g., dietary and antioxidantsupplements). The method comprises contacting the cells with HEDS andsubsequently measuring the amount of extracellular ME. In a particularembodiment, the method comprises providing at least two populations ofcells wherein one population of cells has been contacted with the atleast one compound and a second population of cells as a control whichhas not been contacted with the at least one compound. The methodfurther comprises contacting these populations of cells with HEDS andsubsequently measuring the amount of extracellular ME. An increase inthe extracellular ME from the population of cells contacted with the atleast compound indicates that the at least one compound possessantioxidant properties. In a preferred embodiment, the method comprisesobtaining a biological sample comprising cells from a subject,administering at least one compound to the subject, obtaining a secondbiological sample from the subject, and determining the amount ofextracellular ME after HEDS administration. An increase in theextracellular ME from the second biological sample indicates that the atleast one compound is an antioxidant.

According to another aspect of the instant invention, methods ofscreening tumor cells resistance to chemotherapeutic agents andradiation are provided. In one embodiment, the methods comprise a)contacting a first tumor cell with hydroxyethyldisulfide (HEDS); b)determining the amount of extracellular mercaptoethanol produced by thefirst tumor cell; c) contacting a second tumor cell withhydroxyethyldisulfide (HEDS); and d) determining the amount ofextracellular mercaptoethanol produced by the second tumor cell; whereinthe presence of more extracellular mercaptoethanol from the first orsecond cell indicates that the tumor cell with higher capacity toconvert hydroxyethyldisulfide (HEDS) into mercaptoethanol isresistant/less responsive to cancer therapy. In another embodiment, thefirst and second tumor cells are from biological samples from a subject.Additionally, while the instant methods comprise obtaining a first andsecond biological sample, the instant invention also encompasses amethod wherein only a first tumor sample is obtained and the amount ofextracellular ME after HEDS administration is compared to a standard(s)(e.g., the capacity of other tumor cells and, optionally, non-tumorcells) to convert HEDS and their known responsiveness (e.g., ability totreat, effectiveness of treatment, and/or dosage requirement) to aparticular cancer therapy.

According to another aspect of the instant invention, methods ofscreening blood antioxidant capacity to predict response of a subject tochemotherapeutic agents and/or radiation are provided.

In one embodiment, the methods comprise a) contacting a first bloodsample with hydroxyethyldisulfide (HEDS); b) determining the amount ofextracellular mercaptoethanol produced by the first blood sample; c)contacting a second blood sample with hydroxyethyldisulfide (HEDS); andd) determining the amount of extracellular mercaptoethanol produced bythe second blood sample; wherein the presence of more extracellularmercaptoethanol from the first or second blood sample indicates that thesubject with higher capacity to convert hydroxyethyldisulfide (HEDS)into mercaptoethanol is resistant/less responsive to cancer therapy. Inanother embodiment, the first and second blood sample may consist ofwhole blood, individual blood cells, or serum from a subject.Additionally, while the instant methods comprise obtaining a first andsecond blood sample, the instant invention also encompasses a methodwherein only a first blood sample is obtained and the amount ofextracellular ME after HEDS administration is compared to a standard(s)(e.g., the capacity of other blood samples (e.g., blood samples fromthose undergoing treatment) to convert HEDS and their knownresponsiveness (e.g., ability to treat, effectiveness of treatment,and/or dosage requirement) to a particular cancer therapy).

According to yet another aspect of the instant invention, methods ofscreening blood for HEDS bioreductive capacity to predict the treatmentoutcome of a subject are provided. In a particular embodiment, thesubject has at least one disease or disorder. Exemplary diseases ordisorders include, without limitation, cardiovascular diseases,atherosclerosis, neurological disorders, inflammatory diseases, kidneydisease, adult respiratory distress syndrome, autoimmune disease, liverdisease, Alzheimer's disease, Parkinson's disease, ageing and ageingrelated diseases, allergies, lung disease, diabetes, coronary arterydisease, digestive disease, concussion, over-exercise (exhaustion), druguse (e.g., in athletes), and any disease associated with oxidativestress. In one embodiment, the methods comprise a) contacting a firstblood sample from the subject with hydroxyethyldisulfide (HEDS); b)determining the amount of extracellular mercaptoethanol produced by thefirst blood sample; c) contacting a second blood sample (e.g., from anormal subject) with hydroxyethyldisulfide (HEDS); and d) determiningthe amount of extracellular mercaptoethanol produced by the second bloodsample; wherein the presence of less extracellular mercaptoethanol fromthe first blood sample indicates that the subject with lower capacity toconvert hydroxyethyldisulfide (HEDS) will have poor(er) outcome from thetreatment and may require additional antioxidant therapy. In anotherembodiment, the first and second blood sample may consist of wholeblood, individual (isolated) blood cells, or serum from a subject.Additionally, while the instant methods comprise obtaining a first andsecond blood sample, the instant invention also encompasses a methodwherein only a first blood sample is obtained and the amount ofextracellular ME after HEDS administration is compared to a standard(s)(e.g., the capacity of other blood samples (e.g., blood samples from anormal subject or those undergoing treatment) to convert HEDS andoptionally their known responsiveness (e.g., ability to treat,effectiveness of treatment, and/or dosage requirement) to a particulartherapy).

According to still another aspect of the instant invention, methods ofscreening blood for HEDS bioreductive capacity to predict thesusceptibility of a subject to at least one disease or disorder areprovided. Exemplary diseases or disorders include, without limitation,cardiovascular diseases, atherosclerosis, neurological disorders,inflammatory diseases, kidney disease, adult respiratory distresssyndrome, autoimmune disease, liver disease, Alzheimer's disease,Parkinson's disease, ageing and ageing related diseases, allergies, lungdisease, diabetes, coronary artery disease, digestive disease,concussion, over-exercise (exhaustion), drug use (e.g., in athletes),and any disease associated with oxidative stress. In one embodiment, themethods comprise a) contacting a first blood sample from the subjectwith hydroxyethyldisulfide (HEDS); b) determining the amount ofextracellular mercaptoethanol produced by the first blood sample; c)contacting a second blood sample (e.g., from a normal subject, a subjectwith said disease, or subject known to be at risk for said disease) withhydroxyethyldisulfide (HEDS); and d) determining the amount ofextracellular mercaptoethanol produced by the second blood sample;wherein the presence of less extracellular mercaptoethanol from thefirst or second blood sample indicates that the subject with lowercapacity to convert hydroxyethyldisulfide (HEDS) into mercaptoehtanol issusceptible to disease. In another embodiment, the first and secondblood sample may consist of whole blood, individual (isolated) bloodcells, or serum from a subject. Additionally, while the instant methodscomprise obtaining a first and second blood sample, the instantinvention also encompasses a method wherein only a first blood sample isobtained and the amount of extracellular ME after HEDS administration iscompared to a standard(s) (e.g., the capacity of other blood samples(e.g., blood samples from a normal subject, a subject with said diseaseor a subject known to be at risk for said disease) to convert HEDS andoptionally their known risk for developing said disease).

The instant invention also encompasses diagnostic/prognostic methods. Ina particular method, the method comprises determining the responsivenessof a tumor to a particular treatment and/or determining the response ofa subject to a particular treatment (e.g., chemotherapy and/orradiation) as described hereinabove. Upon determining the responsivenessof a tumor and/or the impact on a subject, the practitioner candetermine whether the particular treatment is to be administered to thepatient or whether a new type of treatment should be sought. Thepractitioner may also determine the dosing level of the treatment neededto treat the disorder (e.g., cancer), while minimizing any negativeimpact on the patient. For example, if it is determined that the tumorwould not be responsive to the treatment, then a new treatment may besought or higher doses may be required/tested. If it is determined thatthe subject would be adversely affected by the treatment, then a newtreatment may be sought or a lower dosage of the treatment may beadministered.

DEFINITIONS

As used herein, the term “oxidative stress” refers to the cytotoxiceffects of oxygen radicals (e.g., superoxide anion (O₂ ⁻), nitric oxide,hydroxy radical (OH), and hydrogen peroxide (H₂O₂)), generated, forexample, as byproducts of metabolic processes that utilize molecularoxygen (see e.g., Coyle et al., Science 262:689-695 (1993)). In otherwords, oxidative stress may refer to a loss of redox homeostasis(imbalance) with an excess of reactive oxidative species (ROS) by theprocess of oxidation. Oxidative stress may refer to a state of a cell ortissue of an animal, in vitro or in vivo.

As used herein, the term “antioxidant” refers to compounds thatneutralize the activity of reactive oxygen species or inhibit thecellular damage done by the reactive species or their reactivebyproducts or metabolites. The term “antioxidant” may also refer tocompounds that inhibit, prevent, reduce or ameliorate oxidativereactions. Examples of antioxidants include, without limitation, vitaminE, vitamin C, ascorbyl palmitate, vitamin A, carotenoids, beta carotene,retinoids, xanthophylls, lutein, zeaxanthin, flavones, isoflavones,flavanones, flavonols, catechins, ginkgolides, anthocyanidins,proanthocyanidins, carnosol, carnosic acid, organosulfur compounds,allylcysteine, alliin, allicin, lipoic acid, omega-3 fatty acids,eicosapentaeneoic acid (EPA), docosahexaeneoic acid (DHA), tryptophan,arginine, isothiocyanates, quinones, ubiquinols, butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA), super-oxidedismutase mimetic (SODm), and coenzymes-Q.

The terms “reactive oxygen species,” or “oxidative species,” as usedherein, refer to oxygen derivatives from oxygen metabolism or thetransfer of electrons, resulting in the formation of “free radicals”(e.g., superoxides or hydroxyl radicals).

As used herein, the terms “host,” “subject,” and “patient” refer to anyanimal, including humans.

As used herein, a “biological sample” refers to a sample of biologicalmaterial obtained from a subject, preferably a human subject, includinga tissue, a tissue sample, a cell sample, a tumor sample, and abiological fluid (e.g., blood or urine). Preferably, the biologicalsample is obtained by in the least invasive manner (e.g., a swab orblood draw).

A “carrier” refers to, for example, a diluent, matrix, adjuvant,preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g.,ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80,Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate,phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol),excipient, auxilliary agent or vehicle with which an active agent of thepresent invention can be maintained. Carriers can be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin. Water or aqueous saline solutions and aqueousdextrose and glycerol solutions may be also employed as carriers.

As used herein, “diagnose” refers to assessing, evaluating, and/orprognosing the disease status (progression, regression, stabilization,response to treatment, etc.) in a patient known to have the disease.

As used herein, the term “prognosis” refers to providing informationregarding the impact of the presence of a disease on a subject's futurehealth (e.g., expected morbidity or mortality, the likelihood ofdeveloping disease, and the severity of the disease). In other words,the term “prognosis” refers to providing a prediction of the probablecourse and outcome of the disease and/or the likelihood of recovery fromthe disease and/or responsiveness to a particular treatment.

Assay

In accordance with the instant invention, methods for measuringoxidative stress are provided. In a particular embodiment, the methodcomprises:

a) providing cells;

b) contacting the cells of step a) with HEDS;

c) measuring the amount of extracellular ME,

wherein a the amount of extracellular ME is inversely proportional tothe amount of oxidative stress. The cells of step a) may be obtainedfrom a subject (e.g., a human). In another embodiment, the cells of stepa) are contained within a biological sample, preferably blood, obtainedfrom a subject. In another embodiment, the cells of step a) areestablished normal human or rodent cells or other types of cells withand without oxidative pentose phosphate cycle. In another embodiment,the cells of step a) are established rodent or human cancer cells orcancer cells from other species with and without oxidative pentosephosphate cycle. The cells of the biological sample may be isolatedand/or washed prior to contacting with HEDS.

The method may also comprise comparing the amount of extracellular MEfrom a test sample with at least one standard. For example, the standardmay be the amount of extracellular ME from cells (e.g., from a subject)which is healthy and/or does not have oxidative stress. The standard mayalso be the amount of extracellular ME from cells (e.g., from a subject)which are experiencing oxidative stress.

In a particular embodiment, the methods of the instant inventioncomprise adding HEDS and the ME-detecting reagent, optionally after adesired amount of time, to the cells without harvesting of the cells,washing, or removal of the medium. In another embodiment, HEDS is addedto the cells and the cells are incubated for a desired amount of time(e.g., 0.25, 0.5, 1, 2, 3, or more hours) before at least a portion ofthe extracellular medium is removed and the extracellular ME is measured(e.g., by HPLC) or the ME-detecting reagent is added after processingthe samples with sulfosalicyclic acid (SSA) buffer.

In a particular embodiment, the ME detecting agent is DTNB. In yetanother embodiment, the amount of ME is measured via O.D. (e.g., theO.D. can be read directly on a microtiter plate (MTP) by a MTP reader).The methods of the instant invention may be performed on microtiterplates (e.g., 96, 24, and 6 well plates) or 100 mm plates.

The amount of extracellular ME can be measured by any technique. In aparticular embodiment, the amount of ME is measured by High PerformanceLiquid Chromatography/Electrochemical Detection (HPLC/EC). For example,samples may be analyzed using a HPLC system comprising a C18 column anda mobile phase of 50 mM phosphate, pH 2.7 with octane sulfonic acid(0.05 mM) and 2.2% acetonitrile. In a preferred embodiment, theextracellular ME is measured by a 5,5-dithiobis 2-nitrobenzoic acid(DTNB) assay (see, e.g., Ayene et al. (2002) J. Biol. Chem.,277:9929-35). For example, extracellular media may be mixed with DTNBand then the O.D. may be measured at 412 nm. The concentration may becalculated using an extinction coefficient of 1.36×10⁴ for reduced DTNB.

Unlike the LDH assay described hereinabove, the method of the instantinvention does not require time kinetics as the time for end point isknown and constant. Furthermore, the optical density for 20,000 cellsfor the instant assay without HEDS and measured 144 hours after platingis 0.24, indicating that the background from medium is very low. Incontrast, the optical density for 20,000 cells after incubating thecells with HEDS for 3 hours and measured 144 hours after plating is 3.2,thereby indicating a spread of 2.7. Therefore, the noise to signal ratioof the instant invention is 15, thereby indicating significantsensitivity.

While HEDS is exemplified herein, any disulfide containing compound maybe used in the methods of the instant invention. Disulfide containingcompounds are readily available (see, e.g., Sigma Aldrich 2006-2007catalog). In one embodiment, the disulfide containing compounds aredi-alkyl disulfides (e.g., disulfides of lower alkyls (i.e., containing1-4 carbons) comprising at least one sulfur atom) or di-aryl disulfides,wherein the members of the disulfide can be the same (symmetricaldisulfide) or different (asymmetrical disulfide). In another embodiment,the disulfide containing compounds are disulfides comprising thiamine,such as, without limitation, thiamine disulfide, thiamine propyldisulfide, and thiamine tetrahydrofuryl disulfide. In anotherembodiment, exemplary disulfide containing compounds include, withoutlimitation, hydroxyethyldisulfide (HEDS; a disulfide of mercaptoethanol(ME)), disulfide of mercaptopropionylglycine (MPG), disulfide of MPG anda lower alkyl, disulfide of MPG and ME, disulfide of mesna(2-sulfanylethanesulfonate), disulfide of MPG and mesna, and disulfideof ME and mesna. In a preferred embodiment, the disulfide containingcompound is HEDS.

Kit

Kits for performing the methods of the instant invention are alsoprovided. In a particular embodiment, the kits comprise 1) HEDS and 2)DTNB. In another embodiment, at least one of HEDS and DTNB is containedwithin a composition comprising a carrier. In still another embodiment,the kit comprises a) 5,5-dithiobis 2-nitrobenzoic acid (DTNB); b)hydroxyethyldisulfide (HEDS); and, optionally, c) sulfosalicyclic acidbuffer (SSA). The kits of the instant invention may further comprise atleast one of: a) HEDS buffer; b) DTNB buffer; and c) reaction buffercontaining at least one of glucose-6-phosphate dehydrogenase,thioredoxin reductase, thioredoxin, or other antioxidants. In yetanother embodiment, the kit further comprises at least one of:microtiter plate(s), buffers (e.g., sulfosalicyclic acid buffer (SSA)),tubes, reaction buffer(s) (e.g., reaction buffers containingglucose-6-phophate dehydrogenase, thioredoxin reductase, thioredoxin, orother antioxidants), and instruction material.

The example set forth below is provided to better illustrate certainembodiments of the invention. It is not intended to limit the inventionin any way.

Example

In live cells, HEDS is reduced to mercaptoethanol, a free sulfhydrylcontaining compound, with no toxic effect on cells suggesting a rapidreduction by intracellular antioxidants. The ME produced inside thecells is extruded into the extracellular medium. This conversionmediated by the antioxidant system can be measured in the extracellularmedium without the need for cellular extract. HPLC electrochemicaldetector was used to quantify the conversion of HEDS intomercaptoethanol in mammalian cells in tissue culture. FIG. 1 shows theHPLC peaks obtained from the extracellular medium after 2 hoursincubation of human cells with HEDS. In the absence of HEDS, theextracellular medium did not have any detectable amount ofmercaptoethanol. However, the extracellular medium showed a large HPLCpeak for ME in 2 hours after incubation of HEDS. ThisHPLC/electrochemical detection system is dependent on the GSH recyclingcapacity of mammalian cells. Similar chromatograms were also obtainedfor extracellular medium from rodent cells incubated with and withoutHEDS. These results indicate that the instant assay is specific for theconversion of HEDS into ME and can be used for human, rodent and othercell types. FIG. 2 shows that the HPLC peaks obtained from theextracellular medium of human cells treated with arsenite, which inducesoxidative stress, showed lack of conversion of HEDS intomercaptoethanol.

In order to determine the sensitivity of the instant assay (HEDS/DTNB)in measuring the oxidative stress induced by arsenite, the effect ofmicromolar concentrations of arsenite ranging from 0, 1, 2, 4, 6 and 8μM was tested in human cells. FIG. 3 shows a linear arseniteconcentration dependent inhibition of HEDS conversion measured byHEDS/DTNB assay using the extracellular medium from these cells. Theresults in FIG. 3 demonstrate that this assay is very sensitive inmeasuring the oxidative stress induced by arsenite, even at micromolarconcentrations.

In order to determine whether the oxidative stress measured after 24hours incubation of cells with arsenite serves as an indicator oftoxicity/cell death, cell death was measured by Coulter Counter Analysissix days after the arsenite treatment. The results in FIG. 4 demonstratean arsenite concentration dependent decrease in the survival of humanHCT116 cells incubated with arsenite for 24 hours. The cell growth wasmeasured six days after removing the arsenite from the extracellularmedium by washing the cells three times with fresh growth medium usingthe methods followed by Ayene et al. (J. Biol. Chem. (2002)277:9929-35). However, HEDS conversion (FIG. 3), which showed arseniteconcentration dependent decrease, was measured immediately after 24hours incubation with arsenite. Yet, HEDS conversion correlated withcell death, which was measured 6 days after the same 24 hours arsenitetreatment. These results demonstrate that the novel HEDS conversionassay can also be an earlier predictor of arsenite toxicity in cells.This assay is the first to quantify oxidative stress induced bymicromolar concentrations of arsenite.

In order to determine whether this assay can be used for High ThroughputScreening (HTS) to quantify metabolically active cells, the instantassay was compared with commercially available assays (XTT and WST-1) ina 96 well platform. The results in FIG. 5 demonstrate that the instantassay shows a linear cell concentration dependent response better thanthe XTT and WST-1. The assays for XTT and WST-1 were carried out as perthe manufacturer's instructions. The assays were performed on a humancolon carcinoma cell line (HCT116 cells; FIG. 5A), normal breast cells(MCF10A; FIG. 5B), and human breast cancer cells (MCF7; FIG. 5C).

In order to determine whether the instant assay can be used for HighThroughput Screening (HTS) to quantify the toxicity of cisplatin, theHEDS conversion by human cells five days after overnight incubation ofcells with 0, 10, 20, 30, 40, and 50 μM of cisplatin was measured in a96 well plate. The results in FIG. 6 demonstrate that the instant assaycan be used to measure the toxicity induced by cisplatin in human cellsin a 96 well plate format.

FIGS. 7A-7C show that the assay of the instant invention can be used todetermine antioxidant capacity in any cell types. Indeed, a cell densitydependent linear response for total antioxidant capacity was observedfor T lymphocytes (Jurkat cells (FIG. 7A) and CCRF-CEM cells (FIG. 7B))and glioma cells (T98G cells; FIG. 7C) with coefficient of determinationvalues (R²) ranging from 0.997 to 1.000.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method for measuring oxidative stress in a cell, said methodcomprising: a) contacting said cells with hydroxyethyldisulfide (HEDS);and b) determining the amount of extracellular mercaptoethanol; whereinthe amount of extracellular mercaptoethanol is inversely proportional tothe oxidative stress of said cell.
 2. The method of claim 1, whereinsaid cells are obtained from a subject.
 3. The method of claim 1,further comprising obtaining a biological sample from a subject.
 4. Themethod of claim 3, wherein said biological sample is blood.
 5. Themethod of claim 3, wherein said biological sample comprises tumor cells.6. The method of claim 3, wherein said subject is suspected of havingbeen exposed to a toxin and wherein the presence of oxidative stress isindicative of exposure to said toxin.
 7. The method of claim 6, whereinsaid toxin is an arsenical.
 8. The method of claim 1, wherein saidmethod further comprises comparing the amount of extracellularmercaptoethanol determined in step b) with the amount of extracellularmercaptoethanol determined for a cell without oxidative stress.
 9. Themethod of claim 1, wherein said method further comprises comparing theamount of extracellular mercaptoethanol determined in step b) with theamount of extracellular mercaptoethanol determined for a cell withoxidative stress.
 10. The method of claims 1, wherein step b) comprisescontacting extracellular fluid with 5,5-dithiobis 2-nitrobenzoic acid(DTNB).
 11. A method for determining the glutathione recycling capacityof a cell, said method comprising performing the method of claim 1,wherein the amount of extracellular mercaptoethanol is directlyproportional to the glutathione recycling capacity of said cell. 12.(canceled)
 13. (canceled)
 14. A method of screening for an antioxidant,said method comprising: a) performing the method of claim 1 on a firstcell; b) contacting a second cell with a compound; and c) performing themethod of claim 1 on said second cell; wherein the presence of moreextracellular mercaptoethanol from said second cell indicates that saidcompound is an antioxidant.
 15. The method of claim 14, wherein saidfirst and second cells are the same cell.
 16. A method of screening foran antioxidant, said method comprising: a) obtaining a first biologicalsample from a subject; b) administering at least one compound orcomposition to said subject; c) obtaining a second biological samplefrom said subject after the administration of said compound; d)performing the method of claim 1 on said first and second biologicalsamples; wherein an increase in extracellular mercaptoethanol in saidsecond biological sample compared to said first biological sampleindicates that said compound or composition is an antioxidant.
 17. Amethod of measuring the cytotoxicity of a compound, said methodcomprising: a) contacting cells with said compound; and b) performingthe method of claim 1; wherein a decrease in the amount of extracellularmercaptoethanol for said cells compared to cells not exposed to saidcompound indicates that said compound is cytotoxic.
 18. A method forscreening for a modulator of antioxidant activity involved in cellsurvival, said method comprising: a) contacting said antioxidant with acompound; b) contacting the mixture of step a) withhydroxyethyldisulfide (HEDS); and c) determining the amount ofmercaptoethanol in the mixture of step b); wherein a modulation in theamount of mercaptoethanol compared to amount of mercaptoethanol in theabsence of said compound indicates that said compound is a modulator ofantioxidant activity.
 19. The method of claim 18, wherein saidantioxidant is selected from the group consisting of glucose-6-phosphatedehydrogenase, thioredoxin reductase, and thioredoxin.
 20. A method ofscreening a sample for having an increased risk of the presence of atoxin, said method comprising performing the method of claim 1 on saidsample, wherein a decrease in extracellular mercaptoethanol in saidsample compared to a control sample without said toxin indicates anincreased risk for the presence of said toxin.
 21. The method of claim20, wherein said sample is water or soil.
 22. The method of claim 20,wherein said toxin is selected from the group consisting of anarsenical, radiation, and a chemotherapeutic agent.
 23. A kit formeasuring glutathione recycling and cell survival, said kit comprising:a) 5,5-dithiobis 2-nitrobenzoic acid (DTNB); b) hydroxyethyldisulfide(HEDS); and c) sulfosalicyclic acid buffer (SSA).